Improving the Z3EV promoter system to create the strongest yeast promoter

Summary Promoters for artificial control of gene expression are central tools in genetic engineering. In the budding yeast S. cerevisiae , a variety of constitutive and controllable promoters with different strengths have been constructed using endogenous gene promoters, synthetic transcription factors and their binding sequences, and artificial sequences. However, there have been few attempts to construct the highest-strength promoter in yeast cells. In this study, by incrementally increasing the binding sequences of the synthetic transcription factor Z 3 EV, we were able to construct a promoter (P36) with approximately 1.4 times the strength of the TDH3 promoter. This is stronger than any previously reported promoter. Although the P36 promoter exhibits some leakage in the absence of induction, the expression induction by β-estradiol is maintained. When combined with a multicopy plasmid, it can express up to approximately 50% of total protein as a heterologous protein. This promoter system can be used to gain knowledge about the cell physiology resulting from the ultimate overexpression of excess proteins and is expected to be a useful tool for heterologous protein expression in yeast.